1. Field of the Invention
The present invention relates to novel non-toxigenic strains of Aspergillus including Aspergillus flavus (A. flavus), Aspergillus parasiticus (A. parasiticus), Aspergillus oryzae (A. oryzae), and Aspergillus sojae (A. sojae); agricultural compositions containing non-toxigenic strains of Aspergillus flavus (A. flavus), Aspergillus parasiticus (A. parasiticus), Aspergillus oryzae (A. oryzae), and Aspergillus sojae (A. sojae) on agriculturally acceptable carriers; and to methods for the control of toxin contamination in agricultural commodities using non-toxigenic strains of A. flavus, A. parasiticus, A. oryzae, and A. sojae.
2. Description of the Related Art
Aflatoxins are potent hepatotoxic, carcinogenic compounds produced by A. flavus Link:Fr. and A. parasiticus Speare (CAST, In: Mycotoxins: Economic and Health Risks. Report 116, 99 pp., Council for Agricultural Science and Technology, 137 Lynn Avenue, Ames, IA 50010). Cyclopiazonic acid (CPA) is another potent mycotoxin that is produced by A. flavus, but not by A. parasiticus. When these fungi invade and grow in commodities such as peanuts, corn, cottonseed, and tree nuts, the resulting contamination with the aflatoxins and CPA often makes the commodity unfit for consumption. The United States peanut industry has identified aflatoxin contamination of peanuts as the number one problem for which a solution is needed (Consensus Report of the National Peanut Council Quality Task Force, 1987, National Peanut Council, Alexandria, Va. 22314). Because peanuts are used primarily for food, strict regulatory limits for the amount of aflatoxin allowable in finished peanut products have been established. Although the United States Food and Drug Administration has an action level of 20 ppb of total aflatoxins in food products, international tolerances for aflatoxin are much lower, typically in the range of 0-4 ppb, and are important because U.S. companies compete internationally in the market to export peanuts and peanut products. For this reason the United States peanut industry has a goal to ensure the delivery of aflatoxin-free peanut products by the year 2000. Although aflatoxin contamination of peanuts can occur during postharvest curing and storage, the most significant contamination usually occurs prior to harvest during periods of late-season drought stress as peanuts are maturing. The only known method for controlling preharvest aflatoxin contamination in peanuts is irrigation, an option that is unavailable to the majority of peanut growers.
Cyclopiazonic acid is an indole-tetramic acid that was first isolated from cultures of Penicillium cyclopium Westling in 1968 (Holzapfel, Tetrahedrom, Volume 24, 2101-2119, 1968). CPA is now know to be produced by a variety of fungi including P. patulum, P. viridicatum, P. puberulum, P. crustosum, P. camemberti, A. flavus, A. versicolor and A. oryzae. In addition, CPA has been found as a natural contaminant of corn and peanuts, often occurring together with aflatoxin (Lansden and Davidson, Applied and Environmental Microbiology, Volume 45, 766-769,1983; Urano et al., Journal of AOAC International, Volume 75, 838-841, 1992). It was also implicated as the causative agent in a human intoxication involving consumption of contaminated millet (Rao and Husain, Mycopathologia, Volume 89, 177-180, 1985). With the discovery of CPA production by A. flavus, 54 isolates of A. flavus were investigated for production of CPA and aflatoxin (Gallagher et al., Mycopathologia, Volume 66, 31-36, 1978). It was found that 28 of the 54 (52%) produced CPA whereas only 18 (33%) produced aflatoxin. Regulatory limits for CPA have not been established; however, because of the co-occurrence of aflatoxin and CPA in commodities, efforts to attain biological control of aflatoxin also need to attain control of CPA.
It has been previously found that co-cultivation of either A. parasiticus or A. flavus with species of Penicillium reduce levels of aflatoxin production while co-cultivation of Fusarium species had no such effect (Ehrlich et al., Experiential, Volume 41, 691-693, 1985). These tests did not involve the use of a soil environment. Co-cultivation with A. niger completely eliminated the production of aflatoxin by a culture of A. flavus (Wicklow et al., Phytopathology, Volume 70, 761-764, 1980). This testing was done under laboratory controlled conditions in which the food source involved sterilized corn.
Cotty (U.S. Pat. No. 5,171,86 Dec. 15, 1992 and U.S. Pat. No. 5,294,442 Mar. 15, 1994) discloses a non-toxigenic strain of A. flavus which inhibits aflatoxin production by toxigenic strains. The patent teaches that agricultural commodities inoculated simultaneously with both a non-toxigenic strain and a toxigenic strain produce seed with up to 100-fold less aflatoxin than commodities inoculated with a toxigenic strain alone. The patent only discloses that the patented strain fails to produce aflatoxin. There is no disclosure of its lack of ability to produce other toxins such as, for example, CPA.
Cole et al.(U.S. Pat. No. 5,292,661 Mar. 8, 1994) and Dorner et al.(Journal of Food Protection, Volume 55, 888-892, 1992) disclose a non-aflatoxigenic strain of A. parasiticus. The references teach the use of this strain as a biocontrol agent which reduces aflatoxin contamination of soil-borne crops.
Tantaoui-Elaraki (Journal of Environmental Pathology, Toxicology and Oncology, Volume 11 (2), 97-101, 1992), Lemke et al. (Applied and Environmental Microbiology, July, 1989, 1808-1810), Jishen et al. (Acta Academiae Medicinae, Volume 8, (1),70-71, February 1986), and Lee (Mycopathologia, Volume 107, 127-130, 1989), all disclose non-toxigenic strains of A. flavus wherein the strains do not produce aflatoxin.
Horn et al. (Mycologia, Volume 88 (4), 574-587, 1996) discloses isolates of A. flavus that fail to produce the mycotoxins CPA and aflatoxin.
While various strains of non-toxigenic Aspergillus for control of toxigenic fungi are known in the art, there still remains a need for an effective biocontrol agent for toxigenic fungi. The present invention described below includes non-toxigenic strains of Aspergillus, especially non-toxigenic strains of A. flavus, A. parasiticus, A. oryzae, and A. sojae, which are antagonistic to toxigenic fungi. The present invention also provides a method for controlling toxigenic fungi in agricultural crops which is different from the related art biocontrol agents.